Chlamydia trachomatis is the cause of trachoma and a variety of sexually transmitted diseases and is the most prevalent reported infectious disease in the United States. Chlamydia are very distantly related to other eubacteria and they have one of the smallest bacterial genomes--1.05 Mb. The entirety of the sequence is being determined using high-throughput technologies as a collaborative effort among investigators at UC Berkeley and Stanford. The availability of the complete C. trachomatis genome sequence mitigates many of the technical research challenges with chlamydiae because this information can be used to direct experimental investigations. Genome sequence information provides a timely and unique opportunity to focus and direct productive and cost-efficient investigation of chlamydial biology. Our long-term goal is to understand the microbiology of this unusual organism sufficiently to use the acquired information for new therapeutic and diagnostic strategies. We propose that virulence and pathogenicity determinants of C. trachomatis can now be effectively investigate experimentally based upon information provided by the complete genome sequence. The specific aims of this application focus on three fundamental properties of chlamydiae that play vital roles for understanding the unique biology of these organisms. The specific aims are: I. Determine the genetic basis of chlamydial developmental regulation. We will determine the genetic basis of developmental regulation by characterizing the time of transcription of each chlamydial gene, produce a comprehensive transcriptional map of the genome, and elucidate the mechanism of transcriptional regulation by chlamydial RNA polymerase sigma subunits. II. identify and characterize environmental signal-transduction response regulators. We will identify sensor-response regulators by homology to those in other eubacteria and characterize their function by in-vitro testing of EB and RB preparations and complementation in E. coli. III. Identify and characterize novel surface and outer membrane components. The surface architecture and outer membrane composition will be approached based upon genome sequence information and the selection of recombinant antibodies to surface components. This will allow analyses of EB molecular content and structure, secretion systems, and host cell ligands that play vital roles in chlamydial structure, virulence and pathogenicity.